The HIV-positive population is at increased risk for mucosal candidiasis and, in late stage AIDS, for disseminated candidiasis. Mirroring what is seen in the HIV positive population for vaginal and esophageal candidiasis, clinical series show that C. glabrata now accounts for about 20% of disseminated candidiasis. The objective of this continuation is to understand the regulation of virulence genes important in disseminated infections. C. glabrata encodes a large number of cell wall proteins in the subtelomeric regions of the chromosome, some of which we have demonstrated to be important in virulence. The subtelomeric regions are poorly described, however, due to difficulties in assembling subtelomeric regions from shotgun sequence. We propose to clone and sequence individual subtelomeres to identify the full complement of genes encoded there; we hypothesize that the annotated subtelomeric pseudogenes are in fact functional and contribute to virulence. We have previously shown that the subtelomeric regions are subject to transcriptional silencing and we have genetically defined many components required for silencing. We propose to generate functional tagged versions of the key silencing proteins and use these to probe the silent chromatin structure using ChIP. Silent adhesin genes are transcriptionally de-repressed in response to environmental signals; we will use chromatin immunoprecipitation to identify the structural changes in chromatin that occur in response to those physiological signals. We have screened a set of 182 C. glabrata strains deleted for orthologues or paralogues of non- essential S. cerevisiae transcription factors. We have identified two regulatory mechanisms important in virulence. Deletions of the Ssn2/Ssn3/Ssn8/Srb8 module of Mediator results in a 10-fold increase in virulence, and deletion of Imp2', a poorly understood regulator of stress genes in S. cerevisiae, profoundly decreases virulence in C. glabrata. We will identify the regulated genes downstream of these two regulators. Our preliminary evidence suggests that sub-telomeric genes, subject to chromatin based silencing, are key targets of both regulators. We hypothesize that they affect infection-specific transcription by altering the phosphorylation status of the RNA polymerase II C-terminal domain (CTD). This grant builds on our previous efforts, which have been uniquely focused on virulence strategies of C. glabrata. Analysis of transcription factors regulating virulence-associated traits has been highly productive in the study of C. albicans, and we believe that our proposed studies, jointly focused on transcriptional regulation of subtelomeric genes will shed new light on virulence strategies in C. glabrata. PUBLIC HEALTH RELEVANCE: The ability of Candida glabrata, an important yeast pathogen, to cause disease is not well understood. We are studying the regulation of virulence genes encoded in the subtelomeric regions of the genome. We have identified two key transcriptional regulators that dramatically alter virulence and are studying how they control the infectious process.